Clément Jimenez

Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study

OBJECTIVE To assess the value of sperm DNA fragmentation, measured by the sperm chromatin dispersion (SCD) test, in predicting fertilization rate, embryo quality, and pregnancy outcome. DESIGN Prospective study. SETTING Four French infertility centers, from January to August 2005. PATIENT(S) Six hundred twenty-two couples participating in their first IVF or ICSI program. INTERVENTION(S) Analysis of DNA fragmentation by the sperm chromatin dispersion test in sperm samples used for IVF or ICSI. MAIN OUTCOME MEASURE(S) Correlations and associations between sperm parameters, sperm DNA integrity, and pregnancy outcomes. RESULT(S) A statistically significant correlation was observed between sperm DNA fragmentation rate and the following sperm characteristics: sperm motility, morphology, and concentration. We found a statistically significant relationship between sperm DNA fragmentation rate and fertilization rate, and we were able to suggest a threshold sperm DNA fragmentation rate of 18%, above which fragmentation rate was predictive of fertilization rate. Regarding embryo quality, we observed a relationship between sperm DNA fragmentation and embryo quality. No significant relationship was found between sperm DNA fragmentation rate and clinical pregnancies or births. CONCLUSION(S) The results of this study confirm the utility of the sperm chromatin dispersion test for assessment of DNA fragmentation.

Phospholipid transfer protein deficiency reduces sperm motility and impairs fertility of mouse males

Vitamin E was discovered for its implication in reproductive biology, and its transport in mammalian plasma and brain was shown to be governed by plasma phospholipid transfer protein (PLTP). We show that PLTP deficiency is associated with hypofertility of mouse males but not mouse females, and it accounts for a significant decrease in total number of pups produced over a 2‐month breeding period of PLTP knocked out mice (−32%, P<0.03). PLTP is highly expressed in epididymis of mouse males, and α‐tocopherol, the main vitamin E isomer in vivo, was significantly less abundant in cauda and caput epididymis of PLTP‐deficient mice as compared with wild‐type counterparts (caput: −26%, P<0.05; cauda: −21%, P<0.05). Mature spermatozoa from PLTP‐deficient epididymis were shown to retain an abnormal α‐tocopherol content. PLTP deficiency tended to reduce sperm motility as shown by a 24% reduction in spermatozoa with progressive motility (P<0.02), with no change in other sperm parameters as compared with wild‐type males. Finally, in vitro fertilization rates of wild‐type oocytes with spermatozoa from PLTP‐deficient males were markedly reduced as compared with those measured with spermatozoa from wild‐type males (−60%, P<0.05). It is concluded that PLTP is a new, key factor that determines sperm motility and male fertility.

Birth after intracytoplasmic injection of epididymal sperm from a man with congenital bilateral absence of the vas deferens who had a robertsonian translocation

OBJECTIVE To present the first case of an infertile male with azoospermia related to a congenital bilateral absence of the vas deferens (CBAVD), in which mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene coexist with a robertsonian translocation. DESIGN Case report. SETTINGS A university hospital. PATIENT(S) A 34-year-old male with a 2-year history of primary infertility. INTERVENTION(S) Lymphocytic karyotype, polymerase chain reaction (PCR) and allele oligonucleotide-specific hybridization (ASO), microsurgical epididymal sperm aspiration (MESA), and intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S) Physical examination and semen analysis. RESULT(S) Semen analyses revealed azoospermia and a well-recognized obstructive phenotype. Analysis of the CFTR gene revealed a compound heterozygosity for a 2184 del A + 2183 A --> G mutation on one allele and the 5T variant within the polypyrimidine tract of intron 8 on the other allele. Cytogenetic analyses revealed a t(13;14)(q10;q10) robertsonian translocation in the same patient. Microsurgical epididymal sperm aspiration allowed retrieval of a million mature motile spermatozoa, excluding any spermatogenic impairment secondary to the genetic abnormalities found in this patient. Epididymal sperm was used for an intracytoplasmic sperm injection program, and a normal child was born at term. CONCLUSION(S) This case illustrates that two distinct genetic defects may coexist, with a variable effect on male fertility but with important implications for genetic counseling of the future pregnancy.

Does Embryo Culture Medium Influence the Health and Development of Children Born after In Vitro Fertilization

In animal studies, extensive data revealed the influence of culture medium on embryonic development, foetal growth and the behaviour of offspring. However, this impact has never been investigated in humans. For the first time, we investigated in depth the effects of embryo culture media on health, growth and development of infants conceived by In Vitro Fertilization until the age of 5 years old. This single-centre cohort study was based on an earlier randomized study. During six months, in vitro fertilization attempts (No. 371) were randomized according to two media (Single Step Medium—SSM group) or Global medium (Global group). This randomized study was stopped prematurely as significantly lower pregnancy and implantation rates were observed in the SSM group. Singletons (No. 73) conceived in the randomized study were included (42 for Global and 31 for SSM). The medical data for gestational, neonatal and early childhood periods were extracted from medical records and parental interviews (256 variables recorded). The developmental profiles of the children in eight domains (social, self-help, gross motor, fine motor, expressive language, language comprehension, letter knowledge and number knowledge – 270 items) were compared in relation to the culture medium. The delivery rate was significantly lower in the SSM group than in the Global group (p<0.05). The culture medium had no significant effect on birthweight, risk of malformation (minor and major), growth and the frequency of medical concerns. However, the children of the Global group were less likely than those of the SSM group to show developmental problems (p = 0.002), irrespective of the different domains. In conclusion, our findings showed that the embryo culture medium may have an impact on further development.

Spermiogenèse : l’acétylation des histones déclenche la reprogrammation du génome mâle

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

La contraception masculine

ResumeDes méthodes de contraception masculine sont actuellement utilisables, en particulier les méthodes mécaniques comme le préservatif, dont le taux d’échec est important, ou la vasectomie qui présente un caractère irréversible. De plus, ces méthodes ne sont pas acceptées par tous les couples.Les méthodes hormonales sont basées sur la suppression réversible de la sécrétion des gonadotrophines (LH et FSH) et sur l’inhibition des stéroïdes intra-testiculaires et de la production des spermatozoïdes. En 1990 et 1996, l’OMS a publié les résultats de deux études qui montrent que la testostérone administrée par injection peut être utilisée comme moyen de contraception. Ces études démontrent en effet pour la première fois que si une méthode hormonale est en mesure de provoquer une azoospermie ou à défaut une oligozoospermie sévère, elle représente une méthode contraceptive efficace.Une autre approche possible consiste à utiliser une combinaison de progestatifs ou d’autres hormones capables de supprimer la sécrétion des gonadotrophines avec les androgènes. L’association permet une meilleure suppression de la spermatogenèse. Les quantités d’androgènes administrées peuvent ainsi être réduites pour diminuer leurs effets indésirables à long terme. Les études sont en cours pour déterminer quelles sont les associations les plus efficaces et les moins dangereuses.Les antagonistes du GnRH permettent d’obtenir la suppression de l’action du GnRH et ainsi de bloquer la spermatogenèse.Les agents agissant directement sur la spermatogenèse sont souvent très toxiques avec une irréversibilité de l’atteinte de la spermatogenèse qui les rend difficilement utilisables dans le cadre d’une contraception.La vaccination contraceptive et, en particulier, l’immunisation avec des protéines impliquées dans l’interaction gamétique, constitue une approche très attractive. Une telle idée n’est pas nouvelle; en effet, plusieurs essais d’immunocontraception, utilisant des modèles animaux ont été rapportés ces dernières années. Toutefois, les résultats de ces études restent plutôt décevants. Cela peut s’expliquer d’abord parce que l’immunoneutralisation d’une seule protéine semble insuffisante pour permettre une diminution significative de la fertilité, et ensuite parce que des taux d’anticorps élevés dans le sérum ne sont corrélés ni aux taux observés dans le tractus génital ni à l’efficacité contraceptive.AbstractCurrently available methods of male contraception include condoms and vasectomy, but condoms have a high failure rate and vasectomy is an irreversible method. These methods are also not accepted by all couples.Hormonal methods are based on reversible suppression of gonadotrophin (both LH and FSH) and inhibition of intra-testicular steroid and sperm production. In 1990 and 1996, the WHO published results from two studies using testosterone injections as a method of hormonal contraception. These studies demonstrated, for the first time, that if a hormonal method is able to induce azoospermia or at least severe oligozoospermia, it could constitute an effective method of contraception.Another possible approach consists of using a combination of progestins or other gonadotropin inhibitors together with androgens to ensure more effective suppression of spermatogenesis. The dose of androgens can be lowered to decrease the risk of long-term adverse effects. Ongoing studies are designed to determine the safest and most effective combinations of androgens and progestins. GnRH antagonists interfere with the action of GnRH and suppress gonadotropins and therefore spermatogenesis.Agents acting directly on the testis are often very toxic and frequently induce irreversible effects on spermatogenesis and therefore cannot be used for contraception.Immunocontraception, particularly targeting of antibodies to gamete-specific antigens involved in sperm-egg binding and fertilisation, constitutes a very attractive approach. This is not a new idea, as several immunocontraception trials, using animal model systems, have been reported over recent years. However, the results of these studies have been largely disappointing because immunoneutralisation of a single, gamete-specific antigen appears to be insufficient to induce a significant reduction in fertility and secondly, although systemic immunisation regimes may lead to high serum antibody levels, these levels do not correlate with specific antibody levels in the reproductive tract or with contraceptive efficacy.

Validation de la mesure d’un paramètre critique en culture d’embryons humains : le pH

In vitro human embryos culture depends largely on the atmospheric conditions within the incubators of the laboratory. The pH of culture media, an indirect reflection of the CO2 content inside these incubators, is a critical parameter. Collaboration between the biochemistry and reproductive biology departments enabled the automated measurement of the pH in the culture medium on a blood gas analyzer. This method has been validated and evaluated. It is applicable in all laboratories whatever the medium and the conditions of culture. It allows strict monitoring of this parameter for the optimization of the culture conditions necessary to improve the results of in vitro fertilization attempts.

Mutations du gène de la polymérase gamma de l’ADN mitochondrial (POLG) associées à l’infertilité masculine?

ResumeLe gènePOLG, localisé en 15q24–15q26, code pour la sous-unité catalytique de l’ADN polymérase mitochondriale γ. La partie N terminale contient une série de 13 glutamines dont les 10 premières sont codées par une répétition du trinucléotide CAG. Cette répétition de CAG est polymorphique, avec un allèle majoritaire de 10 CAG (présent dans 87% des cas dans la population générale). Rovio et al. ont comparé le nombre de répétitions des triplets CAG dans une population d’hommes «contrôle» (population générale), d’hommes fertiles et différentes populations d’hommes infertiles; ils ont observé une association entre l’absence de l’allèle normal (correspondant aux 10 répétitions) et l’infertilité masculine, à l’exclusion de l’azoospermie.Nous avons étudié 503 patients infertiles (dont 84 porteurs d’une microdélétion du chromosome Y) et 90 témoins fertiles et normospermiques. La région contenant les triplets CAG du gènePOLG a été amplifiée et l’analyse de la taille des fragments a été réalisée sur séquenceur.L’allèle prédominant de 10 répétitions de CAG a été retrouvé chez 98,9% des sujets normospermiques et fertiles de façon homozygote ou hétérozygote (génotype 10/10 ou 10/x). Parmi les sujets infertiles, nous avons trouvé 14 patients (3%) ayant à l’état homozygote un nombre de répétitions différent de 10: dans 6 cas, les sujets ont deux allèles mutants identiques (x/x), et dans les 8 autres cas, les sujets ont 2 allèles différents (x/y).Contrairement à l’étude de Rovio et al., parmi les 14 patients homozygotes mutants, deux sont azoospermiques et un est cryptozoospermique. De même, dans notre étude, les sujets infertiles sont aussi souvent hétérozygotes 10/x (26,6%) que les sujets fertiles (27%). De plus, un sujet homozygote mutant 12/12 a obtenu une grossesse spontanée; une seconde grossesse est survenue pendant la prise en charge de son infertilité secondaire. Un autre sujet ayant une mutation a obtenu une grossesse après ICSI, ce qui implique que la mutation n’aurait pas d’effet néfaste sur le développement embryonnaire et l’implantation. A la différence de Rovio et al., un mutant a été trouvé parmi les contrôles fertiles (1,1%).Nos résultats sont les premiers à infirmer ceux de Rovio et al. Ils indiquent qu’il s’agit probablement d’un simple polymorphisme. Il est possible que les résultats présentés précédemment par l’équipe de Rovio et al. proviennent d’un biais d’échantillonnage.AbstractThe single DNA polymerase gene of mitochondrial DNA (POLG) contains a polymorphic CAG repeat in the first coding exon. The promising results of a first study by Rovio et al. showing that several cases of male infertility were related to a “homozygous mutant” genotype with a number of CAG repeats differing from 10, prompted us to conduct a large study on 503 infertile and 90 fertile men.Our results do not confirm any significant relationship between the polymorphic CAG repeat in thePOLG gene and male infertility.